Method of producing l-glutamic acid



nited States This application is a continuation-in-part of our copending application Serial No. 717,841, filed on February 27, 1958, now abandoned, and entitled: Method of Producing L-Glutamic Acid.

The invention relates to a method of increasing the yield of l-glutamic acid (or salt thereof) produced by culturing a microorganism in a culture medium containing carbohydrate, nitrogen source and inorganic material. More specifically the present invention provides a new method for increasing the yield of a compound having an l-glutamate radical by culturing in the presence of an optimum amount of biotin a strain belonging to Micrococcus glutamicus, the concentration of biotin in the culture medium being material.

In co-pending application Serial No. 714,068, filed February 10, 1958, now abandoned, entitled: Method of Producing L-Glutamic Acid by Fermentation, a method is disclosed for producing and accumulating l-glutamic acid in a substantial amount by culturing a strain of Micrococcus glutamicus in a culturing medium.

According to the present invention, the pH control of the fermentation medium set forth in the afore-mentioned application is needed, and it has been found that the yield of l-glutamic acid is remarkably increased if an optimum amount of biotin is present in the culture medium.

It is the primary object of this invention to provide a novel method of producing l-glutamic acid in higher yields than heretofore obtainable. In addition thereto, biotin is efiective for reducing the fermentation period whereby fermentation yield is greatly increased.

According to our investigation, we have also found that there is an optimum range in the amount of biotin present. When the carbohydrate concentration (grams/milliliter) in the medium is from to i.e. weight/volume, the optimum range of biotin is 1 'y/liter. Otherwise the production of l-glutamic acid is greatly reduced. It is noteworthy that biotin has an unique efl ect on l-glutamic acid production.

L-glutamic acid is synthesized through a complicated path from carbohydrate and ammonia. Reasonable fermentation control is especially needed to obtain a high yield of the acid or a salt thereof. In case of this present application, the addition of biotin provides the proper control of the path of glutamic synthesizing reactions.

Investigation was directed to the selection of suitable strains, and it was found that Micrococcus glutamicus No. 534 (ATCC No. 13032) and its mutants, such as Micrococcus glutamicus N0. 541 (ATCC No. 13058), M icrococcus glutamicus No. 5 60, Micrococcus glutamicus No. 582, Micrococcus glutamicus No. 588, Micrococcus glutamicus No. 613 (ATCC No. 13059) and Micrococcus glutamicus No. 614 (ATCC No. 13060), Were suitablefor the above-mentioned purpose. The characters of the above listed strains are tabulated in Table 1.

* atent 2 TABLE 1 Micrococcus glutamicus and its mutants According to Bergeys Manual of Determinative Bacteriology, 6th edition, M. glutamicus is a species of the genus Micrococcus which is most closely related to M. auranziacus and M. epidermidis. A comparison of the three species is set forth in Table 2.

TABLE 2 Comparison of M. glutamicus with M. aurantiacus and M. epidermidis Media M. aurantiacus M. cpz'dermidis M glutamicus Milk Weakly acidic..- Produces acid... N 0 change to slightly alkaline.

Lactose Produces acid do DOGISdDOt produce Nutrient Turbid with Turbid with Slightly turbid,

pellicle. white ring rings are seen and sediment. sometimes along tube. Flocculent sediment. Nutrient agar..- But! to scant White Moderate orange-yellow growth, milky 40 beadttzd wlilite to pale grow ye ow. gglscd, glisteng. Habitat Usually isolated Skin and mu- Air, soil, sewage.

from imeccous memtions but also branes. found in milk, cheese and dust.

As stated in application Serial No. 714,068, referred to supra, page 4, lines 24 to 30, Micrococcus glutamicus is a new species of Micrococcus. This is shown below with reference to the Key to the species of genus Micrococcus as set forth in Bergeys Manual of Determinative Bacteriology.

1. Key to the species of genus Micrccoccus Bergeys Manual, 6th edition:

(1) Aerobic to facultatively anaerobic species.

M. glutamicus is aerobic. (B) Nitrites produced from nitrates.

M. glutamicus produces nitrites from nitrates. (2) Do not utilize NH H PO as a sole source of nitrogen.

M. glutamicus conforms to this description. aa. Gelatin not liquefied or very slowly liquefied.

M. glutamicus conforms to this description. In this section, 2 species namely M. auranziacus and M. epidermidis are included.

from

M. glutamicus 'is compared with and differentiated from M. aurantiacus and M. epidermidis in Table 2. As shown in said table, M. glutamicus is difierent from these two species in pigment on agar, acid production in milk and acid production from lactose.

In addition, in M. epidermidis, Voges-Proskauer reaction is positive although in M. glutamicus, this reaction glatamicus does not utilize NH H PO as a sole source of nitrogen. So, itis clear that M. glutamicus is different from any species listed in Bergeys Manual, 7th edition. That means M. glutamicus is a new species.

M. glutamicus is further distinguished from all the micrococci listed in Bergeys Manual, 6th edition, as set forth in Table 3.

TABLE 3 Comparison of M. glutamicus with other mlcrococcz A B C D E F G H 1. M. 2. M. 3. M. 8. M. 4. M. 5. M. s. M. 7. M. varians. 8. M. caseolyiicua 9a. M. pyogenes var. aureus 9b. M. pyogenes var. albus. 10. M. citreua 11. M. aurantlacus 12. M. epidermidis... 13. M. roseus. 14. M. cinnabareus 15. M. rubens..-- 16. M. rhodochreus... 17. M. agilis 18. M. aerogenes 7 19 M- I. 7 ll 20. M. '21. M. 22.M. anaero ms M glutamicus A-Relation to free oxygen (+:aerobic to facultatlvely anaerobic; anaerobic). ZB-Reddish pigment production on agar.

G-Nitrate reduction to nitrit terlology 6th edition.

Each strain is'compared in the Table 3 (In the absence of other definition the +jandthe indicate the presence and'absence respectively, of the stated characteristic.)

is-negative. This reaction is an important character according to .CJShaw, J. M. Stit and S. T. Cowan (J. Gen. MicrobioLS, 1010, 1951).

11. Key to the genera of family Micrococcaceae from Bergeys Manual, 7th edition, by Breed, Robert 8.; Murray, E.G.D.; and Smith, Nathan R.; published by The Williams & Wilkins Company in Baltimore, Maryland; October 1957:

Therefore, M. glutamicus belongs to genus (1) Micrococcus.

According to the Key to the species of genus Micro- ;coccus in Bergeys Manual, 7th edition:

(1) May or may not reduce nitrates to nitrites. No free nitrogen or nitrous oxide gas produced from nitrates.

M. glutamicus conforms to this key. 'A. Nopink or red pigment produced on agarmedia in young cultures.

M. glutamicus conforms to this key.

(2) Nitrites produced from nitrates.

M. glutamicus produces nitrites from nitrates.

In this section, no species is described which does not utihze NH H PO as a sole sourceofnitrogen. M.

As shown in Table 3,-the species of No. .18 to 22 are fundamentally different 1 from M. glutamicus in their 'anaerobic character. The specicsof No. 13 to No. 17 are definitely difierentiated from M. glutamicus in their red pigment production. The species of No. 1 to No. 5 are clearly diiferent from M. glatamicus in their inability to produce nitrites from nitrates. The species of No. 8 to No. 10 and No. 6 are difierent in gelatin liquefying character and acid production in milk from M. glutamicus. The species of No. 7,.No. 11 and No. 12 are difierent in acid production in milk from M. glutamicus. The species of No. 8, .No. 9a, No. 9b and No. 11 are also difierent follows:

(A) Preparation of l glutamic acid dehydrogenase from micrococci cells The composition of the culture medium was as follows: meat extract 0.5%, peptone 1.0%, glucose 2.0%, NaCl 0.25%, K HPO 0.1%, MgSO -7H O 0.025% and yeast extract 0.2%. The pH of themedium was adjusted at 77.0. Organism tested was :inoculated in the above medium, and cultured for 20 to 24 hours at 28 C. on a shaking-culture device. After the culture, cells were harvested by centrifugation and washed twice with biological saline water. Washed cells were homogenized by grinding with about twice the amount of fine quartz sands. The homogenate thus obtained was centrifuged for 10 minutes at 10,000'r.p;m. The supernatant was in single and irregular masses.

then dialized for 24 hours against M/ 15 phosphate buffer (pH 7.8) at C. This dializate was used as l-glutamic acid dehydrogenase preparation.

(13.) Assay method for the determination of l-glutamic acid dehydrogenase activity In the assay of l-glutamic acid dehydrogenase activity, method of Olson and Antinsen (J. Biol. Chem. 197, 67, 1952), was modified. The composition of the test solution was as follows:

0.5 ml. of M/ 10 l-glutamate solution, 1.0 ml. of coenzyme solution (each I M of TPN and DPN were contained in 1.0 ml. of this solution), and 1.0 ml. of .M/ 15 phosphate buffer, pH 7.8. At zero time, the above test solution and 1.0 ml. of enzyme preparation were mixed in a Beckman spectrophotometric cuvette, and the absorbency at 340 mu was measured every minute for 6 minutes using Beckrnan model DU spectrophotometer. The increase of absorbency during five minutes after the ifirst one minute was defined as AE. AE Was proportional to the concentration of the enzyme if the increment of absorbency was lower than 0.070. Specific activity of the enzyme was defined as AE/ gram of protein in enzyme solution. All the assays were carried out at 30 C.

Results.-The results are shown in Table 4. As seen in the table, Micrococcus glatamicus has considerably higher activity of l-glutarnic acid dehydrogenase than other known species of Micrococcus, therefore M. glutamicus is easily distinguishable from other species.

TABLE 4 Comparative activities of l-glatamic acid dehydrogenase in various species of genus Micrococcus Strain:

Specific activity (AE/gr. of Protein) M. epidermidis ATCC 155 1.4 M. lysodeiticus ATCC 4698 Trace M. varians ATCC 399 6.0 M. citreus ATCC 4012 3.0 M. acesolyticas ATCC 8460 Trace M. soa'onensis Aaronson Trace M. conglomeratas Migula 28.0 M. pyogenes var. albus Schroeter Trace M. flavzts ATCC 10240 75.0 M. glutamicas No. 534 560.0 M. glutamicus No. 541 531.0 M. glatamicas No. 560 343.0 M. glutamicus No. 588 570.0 M. glutamicus No. 582 380.0

The characteristics of Micrococcus glatamicus, repre- 13032), will now be described in detail. The experimental tests were done according to the methods described in the Descriptive Chart of Manual of Methods for Pure Culture Study of American Bacteriologists (9th edition).

Its morphological and physiological characters are as follows.

Slightly ellipsoidal spheres, usually in pairs, but also Size of majority: 0.61.2 microns. Elongated forms are seen in some conditions. Not motile, spores are not formed. Gram-positive.

Agar slant: Moderate growth, filiform, dull, pale yellow.

-: vated, pale yellow.

Broth: Slightly turbid, rings are seen sometimes along mbe. Flocculent sediment, no odor.

Litmus milk: No change or slightly alkaline.

Gelatine stab: No liquefaction or very faint liquefaction.

Hydrogen sulfide not formed. Indole not produced. Starch not hydrolyzed.

Nitrites produced from nitrates. Catalase-positive. Urease produced. Phosphatase-negative. 10 Voges-Proskauer reaction: Negative to very weak.

NH I-I PO is not utilized as a sole source of nitrogen in Huckers medium;

Citrate utilization in Kosers medium is negative. Casein dissimilation: Negative or very weak.

Reduction of dyes: Methylene-blue, 2,6-dichlorophenolindophenol, Janus green and 2,3,5-triphenyltetrazolium chloride are reduced.

Methyl-red test: Weakly acidic. Temperature relations: No growth at 47 C., very slight growth at 42 C., good growth at 28-37 C. Optimum temperature is about 30 C.

Optimum pH, between 7 and 8. Growth occurs between 6 and 9.

Acid from glucose, fructose, mannose, sucrose and maltose. No acid from lactose. (See Table 5.)

-glutamic acid is accumulated in a large quantity aerobically in the presence of carbohydrates, ammonium ion and inorganic salts. Production of organic acid: a-ketoglutaric acid and lactic acid are produced in glucose media.

Aerobic.

Habitat: Air, soil and sewage.

Reference is made to the paper entitled Taxonomical Study of Glutamic Acid Accumulating Bacteria, M icrococcus glatamicas nov. sp. by S. Kinoshita, K. Nakayama and S. Akita. This manuscript was received by the editorial board of Bulletin of the Agricultural Chemical Society of Japan, December 1957 (Faculty of Agriculture, University of Tokyo) 40 Table 5 sets forth the acid production of M. glatamicus No. 534 from carbohydrates.

TABLE 5 Acid production from carbohydrates by Micrococcus glutamicus No. 534

Carbohy- Agar Liquid Carbohy- Agar Liquid drates medium medrates medium medium diurn" Inositol i Melezitose... Arabinose Starch Rhamnose... Xylose :l: Glucose Fructose Ga1actose Mannose Lactose glucoside.

Acid is produced; Acid is not produced; it Acid production is 5 doubtful.

Acid is produced by some other strains oiM. glutomicas. The composition of liquid medium is as follows: peptone 2%, carbohydrate to be tested 1%, distilled water. To 1000 ml. of this solution, 1 ml. of 1.2% bromcresol purple alcohol (95%) solution was added before sterilization. pH of the medium was adjusted to neutrality using N aOH so u on.

In view of the foregoing properties and observations, the inventors recognized that M. glutamicus No. 534 belongs to a new species of the genus Micrococcus. The classification of Micrococcus gluramicus is dependent upon the conditions under which the culture is grown, the

criteria' considered domi antinestablishingthe genus, and

yeast hydrolyzate), digests (e.g. enzymatic digest of yeast) :or the like (e.g. yeast autolyzate, fish solubles and meat scraps). Biotin-containing materials, such as those indicated supra, can be employed in place of pure biotin with substantially equivalent-results. Consequently the present invention is not limited to the use of pure biotin.

Various culturing ingredients described in copending applicationSerial No. 714,068, filed February 10, 1958,

can be used in carrying out the present process.

.In order that the invention may be more clearly understood, practical examples are given below.

EXAMPLE 1 Strain: .Micrococcus glutamicus No. 541 (ATCC No. 13058).

sumo cunmunn Composition of culture medium:

Percent by weight Glucose 2 Famous .7 v 1 Meat extr t 7 0.5 Sodium chloride 0.25 Water The balance. pH, 7 :0-72.

Culturing temperature, 28C.

Procedure: Shaking culture (220 rpm.) with 30 ml.

medium in 250 ml. flasks. Culture duration, 24 hours.

FEBMENTATION CULTURE Composition of culture medium:

Glucose 4)2 4 K HPO z s H 1 MgSO 7H 0 0.25

FeSO -7H O 1 MnS0 -4H O 0.01

Filled up to lliter with water.

Culturing temperature, 28 C.

Biotin was addedto this fermentation medium in various concentrations. 'Micrococcus glutamicus No. 541, isolated by monocolony isolation of Micrococcus glutamicus No. 534, was employed for the instant experiment, but any M icrococcus glutamz'cus, for example one of those micro-organismsspecifically hercinbefore set forth and the mutwt of M icrococcus-glutamicus yield a similar result.

The fermentation medium was subdivided into 30 m1. proportions which were placed into 250 ml. flasks. Sterilization of the medium was carried out in an autoclave at 110 C. for 10 minutes.

3 ml. oiseed culture was added to the sterilized fermentation medium and the submerged culture was done with .a shaking device. Thefermentation was thus carried out underaerobic conditions.

From about 0.5 to about 1.0 .ml. of a 10% (by weight) urea solution was added to the vifv errnentatio'n medium 'at intervals of from about four to about six hours in order to control the pH of said fermentation medium within the range from 6-9.

able 6, show-sthe concentration of l-glutamic' acid in the fermentation medium at stated-intervals after the fermentation was initiated. As is clearly seen from said table, the production of l-glutamic acid was slight and slow in the control experiment but was considerably increased and rapid in the case where 2.57/1. of biotin was added.

TABLE 6 L-glutamic acid concentration (mg/ml.)

Interval after fermentation initiation, hr. 24 48 72 96 Biotin added '(7/] 0 (c trol) 0.2 7.0 16. 5 18.0 1.0 4. 0 18. 0 22.0 '21. 0 r 2.5--- 22. 5 4 41. 8 40:6 6.0.... 18. 5 32. A 31. 6 27. 0 7.5..- 1.8 4. 2 6.0 $1.0 10.0 1. 6 3. G 3.2 .3. 0

:10 gram.

It should be noted further that the production of l-glutamic acid decreased when the amount of added biotin was increased beyond 2.57/1. When Micrococcus glutamicus No. 541 is employed, the optimum range of biotin is from about 1.0 to about 5.07/1.

EXAMPLE 2 The process was identical to that of Example 1 except that Micrococcus glutamicus No. 534 (ATCC No. 13032) was employed rather than Micrococcus No. 541. Where the biotin was added in an amount of 2.57/1. an l-glutamic .acid concentration of 38.2 mg./ml. was obtained after 48 hours culturing.

EXAMPLE 3 EXAMPLE .4

The process was identical to that of Example 1 except 10% (based on weight of fermentation culture) of maltose was added in placeof glucose. Where the biotin was added in an amount of 3.0'y/l., an l-glutamic acid concentration of 31.0 mg/ml. Was .obtained after culturing for 96hours.

smr 5 The process was identical to that of Example 1 except that 1% (based on weight of fermentation culture) of yeast extract was added in place of biotin. After 48 hours cultivation, a concentration of 22.4 mg./ml. was obtained.

EXAMPLE 6 The process was identical to that of Example lvexcept that 1% (based on weight of fermentation culture) .of cane molasses was added instead of biotin. After 48 hours cultivation, a concentration of 24.2 mg./ml. 10f l-glutamic acid was obtained in the fermentation medium.

-C. for 46 hours.

EXAMPLE 7.1,..-

The process was identical to that of Example 1 except that Micrococcus glutamicus No. 614 (ATCC No. 13060, a yellow mutant obtained by subjecting Micrococcus glutamicus No. 534 to ultra violet irradiation using '15 watt U.V. lamp for 5 minutes at the distance of 50 cm.) was employed instead of Micrococcus glutamicus No. 541. When biotin was added to a concentration of 1.0 to 3.07/1, favorable results were obtained, and' at a concentration of 3.0'y/1., 34.3 mg./ml. of l-glutamic acid was obtained in the fermentation medium after 48 hours cultivation.

EXAMPLE 8 Starch acid-hydrolyzate c m1 .1000 (NH4)2HPO4 gram 7 1 (NH4)H2PO4 dO- I 1 MgSO '7H O dO MnSO '4H O ...dO 1 K3504 ..dO-. 1

Starch acid-hydrolyzate has been prepared in the following way: 80 1. of water was added to kg. or pure starch.

added so as to make the total quantity to be 100 l. then neutralized by adding aqueous ammonia, (containing 28.8% of NHs) having the specific gravity of -0.9. This liquid contained 13% of glucose.

EXAMPLE 9 Micrococcus glutamicus No. 534 (ATCC No. 13032) was cultured for 12 hours in a glucose-meat extract-peptone-medium by shaking at 28 C. and used as inoculum for the following experiment.

30 ml. of fermentation medium was added to a 7 -ml. bafiled (Hinton) flask, inoculated with above inoculum, and shaken on a rotary shaker at 220 r.p.m. at 33 The composition of the medium was as follows: 128.0 g. glucose, 5.0 g. urea, 1.0 g. (NH HPO 1.0 g. (NH4)H3PO4, 0.5 g. MgSO 'g. MHSO4'H20, 3.0 g. K 80 2.0 g. (NH SO 0.01 g. FeSO -7H O, 2.5 pg. biotin per liter (water was added to make a total of 1 liter). The medium was sterilized at 120 C. for 5 minutes. The pH was controlled during the fermentation by adding 1 ml. portions of the neutralizing solulions (urea, NH or NaOH) as shown in the below listed experiments. The results are shown in the tables.

EXPERIMENT 1 1 1-29 to l I I EXPERIMENT 111-55" J [Incubation 46-48 hrs. InoculumMlcrococcus glutamicusl I Glutamic Urea NH} N adfi Flask Acid B10 Added} Added, Added) assay,lMg./ percent percent percent m WNSWPFMP-WW moooozooctgw b I l kzo-cnggo- \wa HHIOHHHHMHMNMMHH bald 1 Urea added as 15% solution. NHa added as 14% aqueous solution. NaOH added as 8% solution.

EXPERIMENT III-:60 [Micrococcus glutamtcus] Glutamic Urea Flask Acid Bio Added) Addedfl Added,'. assay,1Mg./ percent percent percent 1 Urea. added as 15% solution. 2 NH; added as 21% aqueous solution. 8 NaOH added as 10.5% aqueous solution.

EXPERIMENT 1 1 1-65.

Glutamic Urea N H; N aOH Flask Acid Bio Added, Added, Added," essay,1Mg./ percent percent percent l Urea added as a 15% solution in flasks 1-4 and as a 30% solution in flasks 5-8 2 NH: added as a 16.8% solution. NaOH added as a 10.6% solution.

3, concentrated sulfuric acid is added until the pH is 3.4. The broth is then coagulated at 87 C. for a period of 5 hours and 2.0 minutes. 40 grams of supercell are added as admixf The slurry containing supercell and broth is then filtered through a No. 4A' porcelain funnel having 40 grams of supercell precoat on it. The filtered cake is washed. with three 125 milliliter portions of water, said water being at a temperature of 80 C. The filtration is rapid.

The filtered broth is then concentrated under reduced pressure to about one quarter of its original volume. The thus concentrated filtered broth is then aged at 5" C.for about 20 hours. During this aging period the broth is agitated.

At the end of the aging period the broth is filtered and (the precipitate washed with four 54 milliliter portions of-water, said water being at a temperature of between to C. The washed precipitate is then airdried, resulting inhigh purity glutamic. acid.

It is thought that the invention and its advantages will be understood from the foregoing description, and. it is apparent that various changes may be made in the process without departing from the spirit and scope. of

the invention or sacrificing its material advantages, the process hereinbeiore. described being merelyv illustrative of' preferred embodiments of the invention.

We claim:

1. In a method for producing. a compoundhaving. an l-glutamate radical from carbohydrate material and nitrogen source by culturing at a pH tromabout 6 to about 9 Mierococcus glutamicus in a culture medium, the improvement wherein said culture medium contains from about 1.0 to about 5.0 otbiotin pe-r'liter.

2. A method of producing ,an l'glutamate selected from thelgroup consisting of. l glutamic acid and salts there of whichfcomprises: (l) culturing Micrococcus glutamicus under aerobic conditions and at a pH" between 6' and about 9, in a liquid culture. medium containing earbohy' drati nitrogen source, inorganic material and from about. liter of said medium and (2) recovering the l-glutamate resultingfrom said c111 1.0 to about 5.0 of biotin per turing.

'3. A method according to claiml wherein the biotin is in the form oia memberselected from the group eons sisting of (a) biotin, b) biotin-containingnaturallsubstance, c.) extract of biotin-containing natural substance,

(d) hydrolyzate of biotin-containing natural. substance, and (e) digest of biotin-containing natural substance.

4. A. method according to claim 1 wherein the biotin is in the form of yeast extract.

5. A method accordingto claim 1 wherein the biotin is inthe 91m of cane molasses.

6". In a; method for producing l-glutamic acid from carbohydrate material and nitrogen source by culturing at a pH; from about 6 to 9 the microorganism correspond ing to ATCC 13059 in a culture medium, the improvement wherein said culture medium contains from about 1.0 to about 5.0 of biotin per liter.

7. In a method for producing l-glutamic acid from carbohydrate material and nitrogen source by culturing at a pH ,from about 6 to about 9 the microorganism corresponding to ATCC 13060 in a culture medium, the improvement wherein said culture medium contains from about -1.0 to about 5.07 oibiotin per liter.

'8. A method of producing l-glutarnic acid which comprises; (1.)- culturing Micrococcus glutamicus under aerobic. conditions in a-liquid culturing medium containing glucose-nitrogen source and from 1.0 to 5.07 per liter of biotin. intheform of a member selected fromthe group consisting of biotin, biotin-containing natural substance, hydrolyzate of biotin-containing natural sub stance, extract of biotin-containing natural substance, and digest of biotin-containing natural substance, the pH of said. culturingmedium being maintained between about 6 and;al 1cu1t 9"v by the. addition thereto of a neutralizing agent, and ('2)? recoveringthe'l-glutamic acid Produced by saidfculturin'g'.

9. In a method carbohydrate material and nitrogen source by aerobically culturing amicroorganism-in aculture medium at a pH from about 6,-to:9, .theimprovement wherein said culture- Inediurn containsv from. about 1.0 to about 5.0 perv liter f biot nt References Citedin the file ot this patent UNITED" STATES PATENTS OTHER REFERENCES Bacterial Chemistry and Physicology by Porter, John Wiley & Sons Inc., New'York'(1946), pages 698 and 699 relied on.

Organic-Chemistry by Fieser et a1., 3rd ed., Reinhold Publishing Corp., New York (1956), page 1010 relied on.

for producing l-glutamic acid from' 

1. IN A METHOD FOR PRODUCING A COMPOUND HAVING AN 1-GLUTAMATE RADICAL FROM CARBOHYDRATE MATERIAL AND NITROGEN SOURCE BY CULTURING AT A PH FROM ABOUT 6 TO ABOUT 9 MICROCOCCUS GLUTAMICUS IN A CULTURE MEDIUM, THE IMPROVEMENT WHEREIN SAID CULTURE MEDIUM CONTAINS FROM ABOUT 1.0 TO ABOUT 5.0Y OF BIOTIN PER LITER. 